Protein kinases play a key role in the regulation of biological systems. The proposed studies focus on (1) structure - function relationships of the cAMP-dependent protein kinases and (2) on the regulation of these kinases by phosphorylation and dephosphorylation. The cAMP-dependent protein kinase is a tetramer composed of 2 regulatory subunits which dimerize and 2 catalytic subunits (R2C2). We have converted the enzyme to a dimer form (RTC) by limited proteolysis. Experiments are proposed to characterize this form in more detail. These studies will be extended to other protein kinase isozymes to establish the minimal requirements for dimerization. Experiments are proposed to isolate other domains of the enzyme, especially the inhibitory domain. The regulatory subunit can be phosphorylated by the catalytic subunit and 2 other kinases -- glycogen synthase kinase 3 and casein kinase 2. The regulatory subunit will be used as a substrate to assay for kinases in tissue extracts in order to establish whether additional kinases for the regulatory subunit exist in heart and other tissues. Such kinases will be purified and characterized. The phosphorylated regulatory subunit will be compared with the dephospho form of the enzyme. Properties which will be examined include subunit-subunit interaction, dissociation constant for cAMP, kinetics for dissociation of cAMP from the two binding sites of the enzyme, and sensitivity to proteolysis. We will study the effect of hormones on phosphorylation of R. This work should lead to a better understanding of both the structure and biological regulation of the cAMP-dependent protein kinases.